We are pursuing the hypothesis that prostate tumors from current, past, and never smokers exhibit differences in their gene expression profiles that are consistent with distinct oncogenic molecular alterations in tumors of current smokers. We are also exploring the effects of nicotine in human prostate cancer cells and TRAMP mice, and are evaluating whether those resemble smoking-associated alterations in prostate tumors. This research is aimed at identifying the mechanisms by which cigarette smoking induces prostate cancer progression, and to define the specific role of nicotine in this process. This proposal combines novelty with a high-impact concept. If we find that nicotine induces disease metastasis, the results could have significant public health implications. Tobacco smoke contains numerous chemicals, including many that are DNA-damaging and carcinogenic. Nitrosamines that are produced from the alkaloid nicotine during post-harvest processing and the burning process of cigarettes are an important group of carcinogens in tobacco smoke. Recently, nicotine and nitrosamines were found to activate signaling pathways in non-neuronal mammalian cells by receptor-mediator mechanisms. Several of these pathways are cancer-related and promote cell survival, angiogenesis, and metastasis. For example, nicotine activates the Akt pathway, which is a key pathway in the development and progression of many cancers, including prostate cancer. In addition, nicotine can reach high nanomolar steady-state concentrations in the blood of current smokers that may activate a signaling pathway in organs other than lung if the appropriate receptors are expressed by the target cells. We collected 67 prostate tumors from 16 current, 28 past, and 23 never smokers for the study, which we obtained from the NCI CPCTR, our resource contract, and Johns Hopkins Medical Institutions. The clinical characteristics of these tumors are similar among current, past, and never smokers. In a pilot, we analyzed the gene expression profiles of tumors from 9 current, 21 past, and 17 never smokers. This analysis revealed a very distinct signature that differentiated tumors from current smokers from those of never and past smokers. Because the first dataset contained tumors from only 9 current smokers, additional prostate tumors were collected at the Department of Urology, Johns Hopkins Medical Institutions (in collaboration with Jun Luo and William Isaacs) and combined with the existing samples to increase the statistical power of our study to identify additional genes that are differentially expressed between current and never/past smokers. The comparison of tumors from current and never smokers yielded 98 transcripts encoding 73 differentially expressed genes. A second comparison, current versus past/never smokers, resulted in a shorter list of only 70 transcripts encoding 40 differentially expressed genes. Likely a residual effect of smoking on the tumor gene signature in past smokers, this study yielded fewer genes when the current and combined past/never smokers were compared. Many of the differentially expressed genes have known immune-regulatory functions. The list also contained interleukin 8 (IL-8), and several others of the differentially genes were found to have an association with hepatocyte growth factor (HGF). The latter is intriguing because both nicotine and HGF activate common pathways, e.g., the PI3 kinase-Akt axis. Some of our observations are preliminary and will need further validation. Nevertheless, the data show that a current smoking status generates a gene signature in prostate tumors that could reveal the mechanism by which smoking causes the metastatic spread of prostate cancer. These observations are being followed up with additional research. We evaluated the relative abundance and expression pattern of IL-8 in prostate tumors from current, former, and never smokers by immunohistochemistry, and also analyzed plasma samples from prostate cancer cases (n = 97) and matched population-based controls (n = 87) with known smoking status to find out whether patients with prostate cancer who are current smokers have higher IL-8 levels in their blood than former or never smokers, or than population-based controls. Immunohistochemistry revealed that IL-8 is expressed by the tumor epithelium and by plasma cell-like immune cells in the prostate of current smokers. The analysis of plasma samples from cases and controls showed that IL-8 is increased in blood samples of prostate cancer patients who are current smokers when compared to former and never smokers among cases and population-based controls. Furthermore, current smokers among population-based controls did not have the same increased IL-8 plasma concentrations that current smoking cases had, indicating that active smoking may lead to increased IL-8 in prostate tumors that can be detected in the blood of these patients. This increased production of IL-8 may also promote the more aggressive behavior of prostate tumors in current smokers, leading to more metastases in this patient group, as found by epidemiological studies. Currently, we are evaluating plasma for lymphotoxin levels to see whether lymphotoxin is increased in the plasma of current smokers who are also prostate cancer patients. We are following up on a recent observation that B cells in prostate tumors can accelerate prostate cancer progression by a lymphotoxin-mediated mechanism. Lastly, we have started to analyze gene expression profiles from the cancerous prostate of TRAMP mice that received physiological concentrations of nicotine for 12 weeks, or tap water only. This experiment is designed to compare the nicotine-induced profiles with the gene expression profiles in prostate tumors from current smokers. It is our hypothesis that these two datasets have a common signature.